U.S. patent number 4,054,646 [Application Number 05/608,255] was granted by the patent office on 1977-10-18 for method and apparatus for detection of antibodies and antigens.
This patent grant is currently assigned to General Electric. Invention is credited to Ivar Giaever.
United States Patent |
4,054,646 |
Giaever |
October 18, 1977 |
Method and apparatus for detection of antibodies and antigens
Abstract
Method and apparatus for the detection of antibodies and
antigens based upon the effect that any arbitrary antigen will
adsorb onto a substrate in a monomolecular layer only, but that a
corresponding specific antibody for such arbitrary antigen will
bond thereto to form a bimolecular layer on the substrate. A first
layer of antigen is adsorbed onto a substrate and the coated
substrate is then exposed to a solution suspected of containing the
specific antibody of interest. The substrate is then examined to
determine whether a monomolecular or bimolecular layer is adhering
thereon. Optical, including visual, electrical, and chemical means
for examining the coated substrate are disclosed. The method can be
reversed by replacing the antigen in each step above with its
specifically reacting antibody and by replacing the antibody with
its specific antigen.
Inventors: |
Giaever; Ivar (Schenectady,
NY) |
Assignee: |
General Electric (Milwaukee,
WI)
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Family
ID: |
27010472 |
Appl.
No.: |
05/608,255 |
Filed: |
August 27, 1975 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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384113 |
Jul 30, 1973 |
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Current U.S.
Class: |
435/5; 427/250;
428/434; 428/469; 435/7.21; 435/7.36; 436/525; 436/806; 436/820;
530/389.4; 530/389.5; 530/806; 600/310; 428/336; 428/478.2;
435/7.31; 436/508; 436/805; 436/818; 530/389.1; 530/413; 530/811;
422/426 |
Current CPC
Class: |
B82Y
5/00 (20130101); B82Y 15/00 (20130101); B82Y
30/00 (20130101); G01N 33/553 (20130101); Y10T
428/31768 (20150401); Y10T 428/265 (20150115); Y10S
530/806 (20130101); Y10S 436/82 (20130101); Y10S
436/805 (20130101); Y10S 530/811 (20130101); Y10S
436/806 (20130101); Y10S 436/818 (20130101) |
Current International
Class: |
G01N
33/551 (20060101); G01N 33/553 (20060101); G01N
021/06 (); G01N 033/16 () |
Field of
Search: |
;23/23B,253TP
;424/12,85,88 ;260/112R |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1,228,698 |
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Apr 1971 |
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UK |
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1,248,764 |
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Oct 1971 |
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UK |
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1,248,765 |
|
Oct 1971 |
|
UK |
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Other References
I Giaever, Jour. of Immunology, vol. 110, 1424-1426 (1973). .
Press Release by General Electric, "Simplified Method of Blood
Analyses Discovered by GE Scientist", issued Nov. 22, 1972. .
A. Rothen et al., Helvetica Chimica Acta, 54, (Fasc. 4), 1208-1217,
(1971). .
Langmuir et al., J.A.C.S., vol. 59, 1406 (1937). .
A. Rothen, Physiological Chem. & Physics, vol. 5, 243-258
(1973). .
Vroman et al., Federation Proceedings, vol. 30, 1494-1502
(1971)..
|
Primary Examiner: Marantz; Sidney
Attorney, Agent or Firm: Morgan, Finnegan, Pine, Foley &
Lee
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part of Ser. No. 384,113
entitled "Improved Method and Apparatus for Detection and
Purification of Proteins and Antibodies" and filed July 30, 1973,
now abandoned.
Claims
I claim:
1. A diagnostic method for determining the presence or absence of a
specific antibody or antigen in a biological sample comprising the
steps of:
depositing a metal onto a substrate material;
contacting said substrate material with an aqueous medium
containing either a corresponding specifically reacting antigen to
said specific antibody or a corresponding specifically reacting
antibody to said specific antigen, to coat all or part of said
substrate material with a monomolecular layer of said specifically
reacting antigen or antibody;
immersing said coated substrate material in said sample; and
examining said coated substrate material to determine whether said
substrate has a bimolecular or monomolecular layer adhering
thereto.
2. The method of claim 1 wherein said step of depositing a metal
comprises evaporating a metal having high atomic mobility and poor
surface wetting characteristics as metal globules onto a light
transmissive substrate material.
3. The method of claim 1 to determine the absence or prsence of a
specific antigen wherein the aqueous medium contains a
corresponding specifically reacting antibody to said specific
antigen.
4. The method of claim 1 wherein said step of depositing a metal
comprises coating said substrate material with a metal film.
5. The method of claim 4 wherein the substrate material is slide
like and also includes a metal oxide layer intermediate the metal
of the slide and said monomolecular layer.
6. The method of claim 5 wherein said metal is titanium and said
metal oxide is an oxide of titanium.
7. The method of claim 1 wherein said step of depositing a metal
comprises evaporating a metal selected from the group consisting of
indium, gold, silver, tin and lead, as metal globules onto a light
transmissive substrate.
8. The method of claim 7 wherein said light transmissive substrate
is selected from the group consisting of glass, plastic, fused
silica, mica and quartz.
9. The method of claim 7 wherein said metal is indium and said
examining step comprises visually examining said substrate and said
determination is made by distinguishing between different shades of
brown.
10. The method of claim 1 wherein said step of depositing a metal
comprises coating said substrate with a thin layer of gold.
11. The method of claim 10 wherein said examining step comprises
visually examining said substrate and said determination is made by
reflectivity measurement.
12. The method of claim 10 wherein another metal is placed between
said gold layer and said substrate.
13. The method of claim 12 wherein said another metal is
indium.
14. The method of claim 1 to determine the absence or presence of a
specific antibody wherein the aqueous medium contains a
corresponding specifically reacting antigen to said specific
antibody.
15. The method of claim 14 wherein said specific antigen is a
protein, contacting is by immersing in said medium and said medium
is a solution.
16. The method of claim 14 wherein said antigen is derived from a
bacteria, a virus, a fungus, mammalian tissue or mammalian body
fluids.
17. The method of claim 16 wherein the antibody is an
immunoglobulin.
18. The method of claim 17 wherein the antibody is of the
immunoglobulin IgG class.
19. A dignostic method for determining the presence or absence of a
specific antibody in a biological sample comprising the steps
of:
coating a metal film onto a substrate material;
contacting said substrate material with an aqueous medium
containing a corresponding specifically reacting antigen to said
specific antibody to coat all or part of said substrate material
with a monomolecular layer of said specifically reacting
antigen;
immersing said coated substrate material in said sample;
placing a mercury drop on the upper surface of said layer on said
substrate material; and
measuring the electrical capacitance between said mercury drop and
said metal coating.
20. The method of claim 19 wherein the antigen is a protein,
contacting is by immersing in said medium and said medium is a
solution.
21. A diagnostic method for determining the presence or absence of
a specific antibody in a biological sample comprising the steps
of:
coating a metal film onto a substrate material, said metal being
capable of forming a visible amalgam with mercury;
contacting said substrate material with an aqueous medium
containing a corresponding specifically reacting antigen to said
specific antibody to coat all or part of said substrate material
with a monomolecular layer of said specifically reacting
antigen;
immersing said coated substrate material in said sample;
placing a mercury drop on the upper surface of said layer on said
substrate material; and
measuring the time elapsing between said placing of a mercury drop
and the appearance of a visible amalgam.
22. The method of claim 21 wherein the antigen is a protein,
contacting is by immersing in said medium and said medium is a
solution.
23. A diagnostic method for determining the presence or absence of
a specific antibody in a biological sample comprising the steps
of:
coating a metal film onto a substrate material;
contacting said substrate material with an aqueous medium
containing a corresponding specifically reacting antigen to said
specific antibody to coat all or part of said substrate material
with a monomolecular layer of said specifically reacting
antigen;
immersing said coated substrate material in said sample;
coating a second metal layer onto the upper surface of said layer
on said substrate material; and
viewing said substrate by reflected light.
24. The method of claim 23 wherein said coating of a second metal
layer comprises electroplating said second metal layer onto said
antigen layer.
25. The method of claim 23 wherein the antigen is a protein,
contacting is by immersing in said medium and said medium is a
solution.
26. A diagnostic method for determining the presence or absence of
a specific antibody in a biological sample comprising the steps
of:
depositing indium as metal globules onto a light transmissive
substrate material by evaporation from a tantalum boat at a vacuum
of approximately 5 .times. 10.sup.-5 mm of mercury;
contacting said substrate material with an aqueous medium
containing a corresponding specifically reacting antigen to said
specific antibody to coat all or part of said substrate material
with a monomolecular layer of said specifically reacting
antigen;
immersing said coated substrate material in said sample; and
examining said coated substrate material to determine whether said
substrate has a bimolecular or monomolecular layer adhering
thereto.
27. The method of claim 26 wherein said antigen is a protein,
contacting is by immersing in said medium and said medium is a
solution.
28. A diagnostic method for determining the presence or absence of
a specific antibody in a biological sample comprising the steps
of:
evaporating a metal having high atomic mobility and poor surface
wetting characteristics as metal globules onto a light transmissive
substrate material;
contacting said substrate material with an aqueous medium
containing a corresponding specifically reacting antigen to said
specific antibody to coat all or part of said substrate material
with a monomolecular layer of said specifically reacting
antigen;
immersing said coated substrate material in said sample;
placing said substrate material, oriented with said metal and said
layer downwards, onto a surface of a light reflecting liquid;
and
examining said substrate to determine whether said substrate has a
bimolecular or monomolecular layer adhering thereto.
29. The method of claim 28 wherein said antigen is a protein,
contacting is by immersing in said medium and said medium is a
solution.
30. A diagnostic method for determining the presence or absence of
a specific antibody in a biological sample comprising the steps
of:
contacting a substrate material with an aqueous medium containing a
corresponding specifically reacting antigen to said specific
antibody to coat all or part of said substrate material with a
monomolecular layer of said specifically reacting antigen;
immersing said coated substrate material in said sample; and
immersing said substrate material in an immunological bond-cleaving
reagent while observing said substrate in an ellipsometer.
31. The method of claim 30 wherein said antigen is a protein,
contacting is by immersing in said medium, said medium is a
solution and said substrate has a surface of a material having a
refractive index substantially different from the refractive index
of said antibody.
32. A diagnostic method for determining the presence or absence of
a specific antibody in a biological sample comprising the steps
of:
contacting a substrate material having a metallic surface with an
aqueous medium containing a corresponding specifically reacting
antigen to said specific protein;
immersing the resulting substrate material in said sample; and
examining said substrate with electrical capacitance sensing means
to determine whether said substrate has a bimolecular or
monomolecular layer adhering thereto.
33. The method of claim 32 wherein said antigen is a protein,
contacting is by immersing in said medium and said medium is a
solution.
34. A diagnostic method for determining the presence or absence of
a specific antibody in a biological sample comprising the steps
of:
contacting a substrate material having a metallic surface with an
aqueous medium containing a corresponding specifically reacting
antigen to said specific antibody to coat all or part of said
substrate with a monomolecular layer of said specifically reacting
antigen, said metallic surface comprising a first electrode;
immersing the resulting substrate material in said sample;
placing a second electrode on the upper surface of said layer;
and
measuring the electrical capacitance between said first and second
electrodes.
35. The method of claim 34 wherein said antigen is a protein,
contacting is by immersing in said medium, said medium is a
solution and placing a second electrode comprises placing a mercury
drop on the upper surface of said layer.
36. A diagnostic method for determining the presence or absence of
a specific antibody in a biological sample comprising the steps
of:
contacting a substrate material having a metallic surface with an
aqueous medium containing a corresponding specifically reacting
antigen to said specific antibody to coat all or part of said
substrate with a monomolecular layer of said specifically reacting
antigen;
immersing the resulting substrate material in said sample; and
examining said substrate employing a chemical sensing means to
determine whether said substrate has a bimolecular or monomolecular
layer adhering thereto.
37. The method of claim 36 wherein said antigen is a protein,
contacting is by immersing in said medium, said medium is a
solution, said metallic surface comprises a metal which forms a
visible amalgam with mercury and said examining step further
comprises the steps of:
placing a mercury drop on the upper surface of said layer; and
measuring the time elapsing between the placing of a mercury drop
and the appearance of a visible amalgam.
38. The method of claim 36 wherein said examining step comprises
immersing said substrate material in a weak acid solution to sever
immunological bonds in said layer while observing said substrate in
an ellipsometer.
39. A surface immunological test method for antibodies to hepatitis
comprising exposing a slide like, metallized substrate having a
monomolecular surface layer comprising hepatitis-associated antigen
over all or part thereof to a solution of an antibody specific to
the hepatitis-associated antigen and producing an immunological
reaction therewith to form a bimolecular layer on the substrate
which can be seen when the substrate is visually examined.
40. A surface immunological test method for antigens to hepatitis
comprising exposing a slide like, metallized substrate having a
monomolecular surface layer comprising antibody to
hepatitis-associated antigens over all or part thereof to a
solution of hepatitis-associated antigen and producing an
immunological reaction therewith to form a bimolecular layer on the
substrate which can be seen when the substrate is visually
examined.
41. A surface immunological test method for gonorrhea comprising
exposing a slide like, metallized substrate having a monomolecular
surface layer comprising Neisseria gonorrhea antigen to a solution
of an antibody specific to the Neisseria gonorrhea antigen and
producing an immunological reaction therewith to form a bimolecular
layer on the substrate which can be seen when the substrate is
visually examined.
42. The method of claim 41 wherein said substrate is a metallized
slide which also includes a metal oxide layer intermediate the
metal of the slide and said monomolecular layer.
43. The method of claim 42 wherein said metal is titanium and said
metal oxide is an oxide of titanium.
44. Medical diagnostic apparatus comprising:
a substrate member;
a plurality of metal globules attached to a surface of said
substrate member; and
a monomolecular layer of a immunologically reactive composition
overlying at least a portion of said substrate and said metal
globules.
45. The apparatus of claim 44 having a plurality of said
monomolecular layers, each said monomolecular layer overlying a
corresponding said portion of said substrate and said metal
globules.
46. The apparatus of claim 44 wherein:
said substrate member comprises a light transmissive wafer; and
said metal globules are indium particles having major dimensions in
the range of 10 A to 2000 A.
47. The apparatus of claim 44 wherein the immunologically
composition is an antibody.
48. The apparatus of claim 44 wherein the immunologically reactive
composition is an antigen.
49. The apparatus of claim 48 wherein said antigen is a
protein.
50. Medical diagnostic apparatus for detecting the presence of an
antibody of interest in a fluid consisting of:
a substrate having a surface including metal; and
a monomolecular layer of a corresponding specifically reacting
antigen to said antibody of interest attached to said surface.
51. Apparatus in accordance with claim 50 wherein said surface
comprises gold, tantalum, indium, titanium or titanium overcoated
with a layer of an oxide of titanium.
52. Apparatus for diagnosing pregnancy in accordance with claim 50
wherein.
said antigen of interest is HCG antigen;
said fluid is urine; and
said specifically reacting antibody is the specifically reacting
antibody for HCG protein.
53. Apparatus for diagnosing hepatitis in accordance with claim 50
wherein:
said antibody of interest is hepatitis antibody;
said fluid is a mixture of blood and hepatitis antibody; and
said specifically reacting antigen is hepatitis-associated
antigen.
54. Apparatus for diagnosing hepatitis in accordance with claim 50
wherein:
said antigen of interest is hepatitis-associated antigen;
said fluid is blood; and
said specifically reacting antibody is hepatitis antibody.
55. Apparatus in accordance with claim 50 wherein said antigen is a
protein, said surface comprises gold and another metal for
improving the adhesion between said gold and said substrate.
56. Apparatus in accordance with claim 55 wherein said other metal
diffuses into said gold to form an alloy thereby increasing the
sensitivity of said apparatus.
57. Apparatus as claimed in claim 50 wherein said surface comprises
a plurality of metal globules attached to said substrate.
58. The apparatus of claim 57 wherein said metal globules consist
of a metal having high atomic mobility and poor surface wetting
characteristics on said substrate.
59. Apparatus for diagnosing gonorrhea in accordance with claim 50
wherein:
said antigen of interest is Neisseria gonorrhea antigen;
said fluid is blood serum; and
said specifically reacting antibody is the antibody to the
Neisseria gonorrhea antigen.
60. Apparatus in accordance with claim 59 wherein said surface
comprises titanium with an overlying layer comprising an oxide of
titanium.
Description
FIELD OF THE INVENTION
This invention relates to an immunological test method and
apparatus for detection of antibodies and antigens. More
particularly, this invention relates to detection of such
biological constituents where the antigen-antibody interaction
takes place at the surface of a substrate.
BACKGROUND OF THE INVENTION
This application contains subject matter which is related to that
in patent application Ser. No. 266,278 entitled "Method and
Apparatus for Detection and Purification of Proteins and
Antibodies" filed June 26, 1972, now abandoned; Ser. No. 392,950
entitled "Method For Binding Antibodies To a Surface Such That They
Remain Active" filed Aug. 30, 1973 now abandoned; and Ser. No.
392,951 entitled "Method for Improving Contrast in Surface
Immunological Tests with Large Size Proteins" filed Aug. 30, 1973,
now abandoned all having a common applicant and assignee.
Other publicantions related to the present invention primarily as
background are "Optical Measurement of the Thickness of a Film
Adsorbed from a Solution", authors Irving Langmuir et al. Journal
of the American Chemical Society, Vol. 59 (July-December 1937) page
1406; "Immunological and Enzymatic Reactions Carried Out at a
Solid-Liquid Interface" by Alexandre Rothen, Physiological
Chemistry and Physics, 5, (1973), pages 243-258; "Interactions
Among Human Blood Proteins at Interfaces", Leo Vroman et al,
Federation Proceedings, Vol. 30, No. 5 (September-October 1971)
pages 1494-1502; and "The Antibody-Antigen Reaction: A Visual
Observation", Ivar Giaever, The Journal of Immunology, Vol. 110,
No. 4 (May 1973) pages 1424-1425.
Immunological reactions are highly specific interactions in which
an antigen interacts with a corresponding second biological
constituent specific to the antigen and generally known as the
antibody to form an immunological complex. Immunological reactions
taking place within a biological system such as an animal or a
human being are vital in combating disease. In a biological system,
the entry of a foreign biological constituent, e.g., the antigen,
causes the biological system to produce the specific antibody to
the antigen in a process not fully understood at this time. The
antibody molecules have available chemical combining or binding
sites which complement those on the antigen molecule so that the
antigen and antibody combine or bond to form the immunological
complex.
Antibodies are produced by biological systems in response to
invasion thereof by foreign bodies. Consequently, the detection of
antibodies present in a biological system is of medical diagnostic
value in determining the antigens to which the system has been
exposed. It would be useful, for example, to test for the presence
of the antibody to syphilis or gonorrhea in human blood, plasma or
tissue. Conversely, the detection of certain antigens in a
biological system also has medical diagnostic values; examples of
diagnostic detection of antigens include detection of human
chorionic gonadotrophin (HCG) protein molecules in urine as a test
for pregnancy, and detection of hepatitis-associated antigen (HAA)
molecules in blood of prospective blood donors.
Most antigens are proteins or contain proteins as an essential
part, whereas all antibodies are proteins. Proteins are large
molecules of high molecular weight, i.e., are polymers consisting
of chains of amino acids. The antigen and antibody protein may each
have several combining sites. The five major classes of antibodies
(immunoglobulins IgG, IgM, IgA, IgE and IgD are each apparently
characterized by at least two heavy (long) peptide chains of amino
acids and at least two light (short) peptide chains of the acids
wherein the bond between the amino acids units is known as a
peptide bond. These heavy and light peptide chains are oriented in
the general shape of the letter "Y" and the active or combining
sites are the extreme ends of the two arms of the Y-shaped antibody
for the IgG antibody.
In addition to the immunological reaction which occurs between
specific protein antigens and specific protein antibodies resulting
in the formation of a protein antigen-protein antibody complex,
other immunological complexing reactions between immunologically
reactive antigens and antibodies are also contemplated by this
invention. In addition, specific reactions between other biological
particles, such as enzymes and their substrates, are also among the
methods contemplated and are embraced by the term "immunological
reaction" as used herein. Furthermore, as used herein, the terms
"antigen" and "antibody" are meant to encompass such terms as
enzymes, substrates, and similar biological particles. As will also
be seen, the method is versatile enough to permit substitution of a
specific antibody for the corresponding antigen and the antigen for
the corresponding specific antibody.
For instance, the following systems include biological particles
which are capable of undergoing the immunological reactions
described herein:
Viruses
Bacteria and Bacterial toxins
Fungi
Parasites
Animal tissue
Animal body fluids, and the like.
With respect to viruses, the antigens are viral cultures, or parts
thereof, and the antibody specific thereto can be produced by
administration to a living host. Illustratively, antigen-antibody
complexes in the following virus systems are useful in the
hereindisclosed procedure: Rubella virus culture (antigen) --
Rubella virus antibody; polio virus culture (antigen) -- polio
virus antibody; vesicular stomatitis virus (VSV) culture (antigen)
-- VSV antibody.
Regarding bacteria and bacterial toxins, the antigens are the
particular bacteria or bacterial toxin, or parts thereof, and the
antibody is produced by injection into a living host. The following
are illustrative examples of antigen-antibody pairs which can be
used in the present method: tetanus toxoid suspension (antigen) --
tetanus antibody; diptheria toxin suspension (antigen) -- diptheria
antibody; Neisseria gonorrhoea suspension (antigen) -- gonorrhea
antibody; Treponema palladium suspension (antigen) -- syphilis
antibody.
As for fungi, the antigens are antigenic extracts of fungal
suspensions and the antibody is the fungal antibody produced by
injection into a living host. Antigen-antibody complexes of fungi
systems are illustrated by the following: Aspergillus extract
suspension (antigen) -- aspergillus fungus antibody; Candida
extract suspension (antigen) -- candida fungus antibody.
Antigens and antibodies in parasite systems are tested in a similar
fashion to those of fungi. The system Toxoplasma gondii extract
(antigen) -- Toxoplasma gondii antibody is a typical example.
By the term polysaccharides is meant a system wherein the antigen
is a carbohydrate antigen. An example of such an antigen-antibody
containing system is pneumococcus polysaccharides (antigen) --
pneumococcus antibody.
In addition to the typical enzyme-enzyme substrate reaction which
is intended to be covered herein, enzymes themselves, or parts
thereof, may be utilized as antigens, and the antibody is the
particular enzyme antibody elaborated by a living host after
injection. Illustrative antigen-antibody complexes of enzyme
systems are:
trypsin extract -- trypsin antibody
chymotrypsin extract -- chymotrypsin antibody
pepsin extract -- pepsin antibody
ribonuclease extract -- ribonuclease antibody
thrombin extract -- thrombin antibody
amylase extract -- amylase antibody
penicillinase extract -- penicillinase antibody
With respect to hormones, the antigenic constituent is usually
found in a hormone extract and the antibody is the particular
hormone antibody elaborated by the living organism after injection.
An exemplary antigen-antibody complex is:
insulin -- insulin antibody.
Although the ensuing discussion is directed for the most part to
immunological interactions between specific protein antigens and
specific protein antibodies, it is understood that it also applied
to the systems and the immunologically reactive antigens and
antibodies hereinabove described.
As presently practiced, the diagnostic utilization of
immunologically active antibodies and antigens relay upon the
precipitating or agglutinating characteristic thereof resulting
from the immunological complexing reaction. The classic example of
these diagnostic uses is the blood typing procedure in which blood
samples are mixed with alpha and beta type serum antibodies and
blood type is determined by observing any agglutination occurring
in the blood samples.
The HCG pregnancy test as currently practiced is an inhibition
test. The test is performed by mixing a quantity of HCG anti-serum
into a urine specimen. A plurality of polystyrene spheres which
have been coated with HCG protein are then introduced into the
previously prepared urine specimen. The polystyrene spheres will
agglutinate if, but only if, HCG protein is absent from the urine
specimen. If HCG protein is absent from a urine specimen, the HCG
protein on the polystyrene spheres complexes with the HCG
anti-serum previously introduced in the urine specimen and the
spheres agglutinate. If, on the other hand, HCG protein is present
in sufficient quantities in the urine specimen, it complexes with
the previously introduced HCG anti-serum forming a complex which
precipitates out of the specimen so that the previously introduced
anti-serum is no longer available to complex with the HCG protein
on the spheres to cause agglutination thereof. In accordance with
the teachings of this disclosure, the present HCG protein pregnancy
test could be simplified by adhering HCG anti-serum onto the
polystyrene spheres and directly testing a urine specimen. In this
case, the polystyrene's spheres would agglutinate if, but only if,
HCG protein is present in the specimen.
It appears that the reason this simpler procedure has not been
employed is that the available HCG anti-sera are complex mixtures
containing a large proportion of constituents other than HCG
antibodies. The additional effort required in the prior art to
extract the antibodies from the HCG anti-sera make the inhibition
test, utilizing sera directly, preferable in the prior art.
However, in accordance with one embodiment of this invention, a
procedure is provided whereby the simpler, direct test is
performable. A serious shortcoming of agglutination tests overcome
by the present direct method is that the particles involved may
tend to agglomerate for any of a variety of reasons having nothing
to do with immunological agglutination thereby decreasing the
reliability of the test. Typically, even though agglutination tests
are performed with great care by skilled technicians, nevertheless
occasional diagnostic errors occur.
Even though it is known that the antibody-antigen complexing
reaction will take place when an antigen (or antibody) is adsorbed
at a surface, the technique has not been widely applied to routine
medical diagnoses on slide like substrates. The complexing reaction
at a surface has been observed by means of an ellipsometer. An
ellipsometer is a complex optical instrument by means of which it
is possible to measure the thicknesses of films on the order of 0.1
A. Ellipsometers are expensive and require skilled operators. In
studies of immunological reactions using ellipsometers performed to
date, two methods have been used. In one method, the reaction to be
studied is allowed to take place and then the slide on which the
reaction has taken place is mounted in an ellipsometer and an
actual measurement of film thickness is made. In the other method,
the slide is mounted in the ellipsometer while the immunological
reaction is taking place and the change of film thickness is
observed with the ellipsometer. The measurement of absolute
thickness requires extreme care. On the other hand, when the
concentration of antibodies in the solution is low, the measurement
of absolute thickness will take a long time. Accordingly, for
practical reasons, the detection of immunological reactions at a
surface using an ellipsometer has not been adopted for diagnostic
purposes.
The present invention is based on the discovery that any arbitrary
immunologically reactive antigen will adsorb onto a substrate in a
monomolecular layer only, and that a specific antibody for such
arbitrary antigen will bond thereto to form a bimolecular layer on
the substrate. In practicing this invention, such layers are easily
and quickly detectable. This discovery then provides methods and
apparatus of great utility in medical diagnostic and
pharmacological applications.
Accordingly, it is a first principal object of this invention to
provide method and apparatus for economically detecting
immunological reactions occurring at a surface.
It is an object of this invention to provide such method and
apparatus wherein such immunological reactions are detectable by
electrical means.
It is another object of this invention to provide such method and
apparatus for detecting such immunological reactions by direct
visual observation.
A second principal object of this invention is to provide method
and apparatus for detecting the presence or absence of antigens and
antibodies by means of controlled immunological reactions occurring
at a surface.
SUMMARY OF THE INVENTION
In accordance with this invention, a diagnostic method is provided
for determining the presence or absence of a specific antibody in a
biological sample comprising the steps of depositing a metal on a
substrate material, contacting the substrte material with an
aqueous medium containing a corresponding specifically reacting
antigen to said specific antibody to coat all or part of the
substrate material with a monomolecular layer of the specifically
reacting antigen, immersing the coated substrate material in the
biological sample, and then examining the coated substrate to
determine whether the substrate has a bimolecular or monomolecular
layer adhering thereto. If a bimolecular layer is detected, the
diagnosis is that the biological sample does, in fact, contain the
specific antibody of interest. Lack of a bimolecular layer is
evidence that the simple does not contain the same.
The novel features of this invention sought to be patented are set
forth with particularity in the appended claims. The invention,
together with further objects and advantages thereof, may be
understood from a reading of the following specification and
appended claims in view of the accompanying drawings wherein like
parts in each of the several figures are identified by the same
reference character.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a flow chart illustrating the process steps of the
various embodiments of this invention;
FIGS. 2a, 2b and 2c are elevation views of diagnostic apparatus in
accordance with this invention illustrating the method of making
and using same;
FIGS. 3a, 3b and 3c are elevation views of apparatus useful for
diagnostic purposes in accordance with another embodiment of this
invention;
FIG. 4 is a photograph illustrating the operation of diagnostic
apparatus in accordance with this invention;
FIGS. 5a and 5b are elevation views of diagnostic apparatus;
FIG. 6 is an elevation view of diagnostic apparatus in accordance
with this invention illustrating a modification in the method of
using the apparatus of FIG. 2;
FIG. 7 is a graphical representation illustrating the principle of
operation of diagnostic apparatus in accordance with the embodiment
of this invention illustrated in FIG. 8;
FIG. 8 is an elevation view of another embodiment of diagnostic
apparatus in accordance with this invention.
DETAILED DESCRIPTION OF THE INVENTION
Referring now to FIG. 1, in reading the flow chart from top to
bottom, each vertical level represents one time sequential step of
the process. The appearance of several steps arrayed horizontally
at a given vertical level in the flow chart indicates the
alternative performance of one of the indicated steps at the
indicated sequential position in accordance with the various
embodiments of this invention.
In accordance with a first embodiment of this invention, the
process begins at block 13 of FIG. 1 in which a wafer of substrate
material which may be metal, glass, mica, plastic, fused silica,
quartz, or similar material, with metal being preferred, as having
the greatest difference in refractive index to the antigen or
antibody, and preferably is in the form of a metallized glass
slide, is contacted with a solution containing a first antigen of
interest which is available in a relatively concentrated aqueous
medium, e.g., a solution, and which is to be used to detect or
purify its corresponding specifically reacting antibody. Contacting
may be by any technique, such as applying one or more drops of
medium or by partial or total immersion. Depending upon the
technique and the concentration thereof, the first antigen adsorbs
onto all or part of the surface of the substrate in a monomolecular
layer. Any antigen will adsorb in such monomolecular layer but no
further adsorption will take place. That is, the antigen will
attach to the substrate, but will not attach to itself. The time
required to adsorb the monomolecular layer on the substrate is a
function of the concentration of the antigen in the solution and
the degree of agitation of the solution. As an example, a 1 percent
bovine serum albumin solution completely coats a slide in
approximately 30 minutes with a monomolecular protein layer. The
next step, illustrated in block 14 of FIG. 1 is to immerse the
antigen coated substrate in a solution suspected of containing the
specifically reacting antibody to the first antigen. This solution
may, and typically does, contain many constituents in addition to
the specifically reacting antibody whose presence it is desired to
detect. However, no antibody other than a specifically reacting
antibody will adhere to the first layer on the substrate. Of
course, as is known, certain biological substances, may adhere by a
phenomenon known as non-specific sticking. Such substances are
found in sera, etc. There are a number of ways to minimize
non-specific sticking, the most convenient of which appears to be
in diluting the media carrying such substances. In any event, if
the corresponding specifically reacting antibody is not present,
the substrate following immersion in the suspected solution will
still contain only a monomolecular antigen layer thereon. If, on
the other hand, the corresponding specifically reacting antibody is
present in the solution, immunological complexing between the first
antigen and its corresponding specifically reacting antibody will
take place and the substrate will, after a time, have a bimolecular
layer thereon. It is to be noted that the steps illustrated in
blocks 13 and 14 of FIG. 1 are common to all of the embodiments of
this invention. The time requirement for the adhesion of a complete
second molecular layer onto the coated substrate is again a
function of the concentration of corresponding specifically
reacting antibody in the solution. For antibodies in blood serum,
this time may be as long as one day, or as short as minutes,
depending on the concentration. In one embodiment of this
invention, the next step is to immerse the coated substrate in a
reagent capable of cleaving immunological bonds as illustrated in
block 15 of FIG. 1 while simultaneously observing the coated
substrate in an ellipsometer as illustrated in block 22 of FIG. 1.
Such a reagent can be a weak acid solution, a base solution or a
strong salt solution, and the like. While the formation of the
specifically reacting antibody on the antigen coated substrate may
take an extended period of time, the immunological bond between the
two layers is severed very quickly by the immunological
bond-cleaving solution. Accordingly, the observation made with the
ellipsometer can be the relatively simple observation of change of
film thickness rather than the more complicated measurement of
absolute thickness. At the same time, observing the stripping away
of the specifically reacting antigen/antibody layer by action of
the immunological bond cleaving solution may be performed much more
rapidly than the prior art method of observing the building of the
specifically reacting antibody layer. Accordingly, a large
plurality of test slides may be prepared in accordance with block
13 and each exposed to one of a large plurality of solution, e.g.,
serum samples as indicated in block 14, and then in a short time
each test slide may be examined serially in accordance with blocks
15 and 22 to determine which of the serum samples contained the
specifically reacting antibody to the first antigen. Since the
immunological bond cleaving reagent will not strip the first
antigen or antibody from the substrate, those coated substrates
which were immersed in media which did not contain the specifically
reacting antibody will exhibit no change of film thickness when
immersed in the cleavage reagent and observed in an ellipsometer.
On the other hand, those which were immersed in a solution which
did contain the specifically reacting antibody will exhibit
approximately a factor of 2-5 change in thickness when immersed in
the cleavage reagent and observed in an ellipsometer. Each
observation can be made in a few minutes thereby providing an
efficient, and therefore diagnostically significant, test
procedure.
While the first described embodiment improves the efficiency of
ellipsometer immunological detection to the point of practical
feasibility, it is nevertheless economically desirable to eliminate
the need for the ellipsometer entirely. Accordingly, another
embodiment of this invention provides for determination of relative
film thickness by electrical means. In this embodiment, either a
metallic substrate is chosen, or a substrate of another material is
first coated with a metal film as indicated in block 11 of FIG. 1.
The metal substrate or metal-coated substrate is then coated with
media containing antigens in accordance with the steps of blocks 13
and 14 as discussed above. The layers adhering to the substrate are
electrically insulating. The next step is to place a mercury drop
or other electrode upon the upper antigen layer adhering to the
substrate as shown in block 16 of FIG. 1. The upper layer is either
the first antigen, if the suspected solution contained no
corresponding specifically reacting antibody, or the specifically
reacting antibody if the solution did contain it. Accordingly, the
metal film or metal substrate and the mercury drop comprise the two
conducting plates of an electric capacitor separated by an
insulating layer comprising either a monomolecular layer or a
bimolecular layer of the antigen or antibodies. The electric
capacitance of this capacitor is then measured as indicated at
block 19 of FIG. 1 by means of any suitable instrument for
measuring capacitance as is known in the art, for example, Heathkit
Impedance Bridge Model IB-28. The electrical capacitance of such
capacitors having a bimolecular layer dielectric is, as expected,
found to vary by approximately a factor of 2-5 from that of such
capacitors having a monomolecular layer.
Another, closely related, embodiment of this invention further
simplifies the detection of the specifically reacting antibody by
enabling its presence to be ascertained by unaided visual
observation. This embodiment employs a substrate of a metal which
forms an amalgam with mercury or another substrate material such as
glass which is coated in the block 11 step with a thin film of
metal which forms an amalgam with mercury, preferably gold. The
substrate is then subjected to the block 13 and block 14 steps as
before and a mercury drop is again placed on the upper layer as
indicated in block 16. In this embodiment, however, no electrical
measurement is made. In this embodiment, the next step is to
visually observe the coated substrate and determine the length of
time which elapses before a visible amalgam is formed between the
mercury and the metal film coated on the substrate. This embodiment
of the invention depends upon the inventive discovery that mercury
diffuses through a monomolecular layer in approximately 1 minute,
but requires 10 minutes or longer to diffuse through a bimolcular
layer. Since the mercury must diffuse through the layer or layers
to form a visible amalgam with the metal film coating on the
substrate, the time between the placing of the mercury drop upon
the upper layer and the appearance of the visible amalgam indicates
whether a monomolecular or bimolecular layer is present on the
substrate and consequently whether or not the suspected solution
actually contained corresponding specifically reacting antibody to
the first antigen. At this point, those skilled in the art will
realize that the capacitance measurement of the last previously
discussed embodiment must be made within 1 minute of the placing of
the mercury drop upon the upper layer lest the mercury electrode
diffuse through the insulating layer and short out the capacitor.
Alternatively, shorting may be prevented by coating the metal with
a metal oxide insulating layer and adhering the antibody or antigen
onto the metal oxide. It will also be recognized that a rough
quantitative measure of the concentration of specifically reacting
antibody in the suspected solution may be made in accordance with
this embodiment by preparing a plurality of substrates in the block
13 step and immersing them simultaneously in the suspected solution
as indicated in block 14, but removing them sequentially from the
solution over an extended period of time. Since the rate at which
the corresponding specifically reacting antibody layer forms is a
function of the concentration of specifically reacting antibody in
the solution, a comparison of diffusion times for the mercury drop
through the layers on a series of substrates having been exposed to
the suspected solution for varying periods of time will provide a
rough quantitative indication of the concentration of specifically
reacting antibody in the suspected solution.
In another related embodiment, a substrate is coated with a metal
film as depicted in block 11 of FIG. 1. As discussed in the last
embodiment, the substrate is then contacted with the media as shown
in blocks 13 and 14. The next step in accordance with this
embodiment is to coat the upper antigen or antibody layer with a
second metal layer as illustrated in block 17 of FIG. 1 and to view
the structure thus produced by reflected light as illustrated in
block 21 of FIG. 1. The second metal is preferably applied by
electroplating. Essentially, this embodiment produces a structure
which functions as a diffraction grating and the difference between
a monomolecular and bimolecular antigen or antibody insulator layer
is determinable from the spectral components observable in the
reflected light.
FIGS. 5a and 5b illustrate apparatus in accordance with this
embodiment. A metallic surface 81 of a substrate which may be
either a metallic substrate or a non-metallic substrate coated with
a metal film, has a first antigen layer 82 applied thereto as
indicated in block 13 of FIG. 1. The substrate having layer 82 is
then immrsed in the solution to be tested and a second layer 83 of
corresponding specifically reacting antibody to the antigen of
layer 82 forms thereon if it is present in the solution. A second
layer of mtallization is then applied to the exposed surface of the
film. A very small quantity of metal is applied so that the second
metallization tends to be in the form of a plurality of
discontinuous metallized areas, as, for example, 84 and 85. A beam
of light represented by rays 86 is then directed onto the slide
thus prepared. The light is reflected at metal boundaries. The
distance between the reflecting surfaces of metal substrate surface
81 and metallization particles, 84, for example, is a function of
the thickness of the film between members 81 and 84. Accordingly,
the observed reflected light, illustrated by rays 87, varies in
spectral composition as the antigen or antibody layer varies
between a monomolecular layer and a bimolecular layer.
Another embodiment of this invention illustrated in FIG. 1
comprises the steps illustrated at blocks 12, 13, 14 and 18 of FIG.
1. In this embodiment, the substrate which must be a light
transmissive substrate such as glass, plastic, fused silica, mica,
quartz, or the like, and is preferably glass, with microscope
slides being a conveniently available source, is first coated with
a plurality of metal globules by evaporating a metal, for example,
indium, onto the substrate as indicated in block 12 of FIG. 1. For
example, the indium is evaporated slowly from a tantalum boat onto
the glass substrate in an ordinary vacuum of about 5 .times.
10.sup.-5 mm of mercury. Because the indium atoms have high
mobility on the surface of the substrate and do not wet the glass
substrate significantly, the indium evaporated onto the substrate
agglomerates into small particles. Any metal having similar
characteristics so that it will form globules on the substrate when
evaporated thereon may be used. In addition to indium, gold,
silver, tin, lead, and bismuth may be used. The evaporation of
metal is continued until the substrate appears light brown in
color. At this point, the metal globules have diameters on the
order of 1000 A. The precise size of the globules is not critical
but they must have diameters equal to a large fraction of the
wavelengths of visible light. The next step is to contact the
globule-covered substrate with a medium containing an antigen as
illustrated in block 13 of FIG. 1. The antigen again adheres in a
monomolecular layer over the substrate and the metal globules
thereon. When a monomolecular layer has formed, the coated
substrate may then be used to test suspected solutions for the
presence of a corresponding specifically reacting antibody to the
antigen by immersing the coated substrate in the suspected solution
as indicated in block 14 of FIG. 1. If the suspected specifically
reacting antibody is present, the substrate and metal globules have
a bimolecular layer adhering threto; if the specifically reacting
antibody is not present, only a monomolecular layer overlies the
substrate and metal globules. The coated substrate is then viewed
by either reflected or transmitted light as indicated in block 18
of FIG. 1 and a determination is made from the appearance of the
coated substrate as to thickness of the layer adhering thereto and
accordingly as to the presence or absence of the suspected
specifically reacting antibody. The detection of layers corresponds
to variations in the shade of brown which is observed in the coated
substrate. These variations are quite pronounced and the detection
of layers is therefore a simple straightforward procedure. The
particles alone on the substrate appear as a first shade of brown,
the particles coated with a monomolecular antigen layer appear as a
darker shade of brown, and the particles covered with a bimolecular
layer appear as a still darker shade of brown. This detection
method is based on the fact that electromagnetic radiation is
scattered to a large degree by conducting spheres having diameters
equal to a large fraction of a wavelength of the incident light and
that in the case of scattering from such spheres, the scattering is
strongly influenced by a thin dielectric coating applied to the
spheres.
FIG. 4 is a reproduction of an actual photograph of diagnostic
apparatus in accordance with this embodiment. In each view of FIG.
4 the test slide is viewed by transmitted light. View 4a is a glass
slide having indium globules over one surface 100 thereof. View 4b
shows a slide prepared as the slide of view 4a whose left edge has
been immersed in a solution of bovine serum albumin, thereby
adsorbing a monomolecular layer 101 of bovine serum albumin
thereon. View 4c shows a similar slide in which the left edge has
been immersed in bovine serum albumin thereby adhering a
monomolecular layer 101 of bovine serum albumin thereon, and whose
lower edge has been immersed in a solution of ovalbumin thereby
adsorbing a monomolecular layer 102 of ovalbumin thereon. It is
important to note that the appearance of the coated portions of the
slide of view 4b are similar, indicating that only a monomolecular
protein layer is present on the slide. This demonstrates that
bovine serum albumin and ovalbumin absorb onto the slide and that
the ovalbumin does not adhere to the portion of the slide
previously coated with bovine serum albumin. In other words, view
4c illustrates the discovery discussed above that any arbitrary
antigen or antibody will adhere to the substrate but another
antigen or antibody will not adhere to an arbitrary antigen or
antibody film, except for immunological complex formation and the
possibility, as noted above, for some non-specific sticking to
occur. The slide of view 4d was prepared by immersing fthe left
edge thereof in bovine serum albumin in solution to adhere
monomolecular layer 101 thereon; the right edge was then immersed
in an ovalbumin solution to adhere monomolecular layer 102 thereon;
and finally the lower edge was immersed in a solution of rabbit
anti-serum to bovine serum albumin. Accordingly, 103 is a
monomolecular layer of rabbit anti-serum to bovine serum albumin.
The appearance of the lower right-hand corner indicates that the
rabbit anti-serum to bovine serum albumin did not adhere to
ovalbumin layer 102 and accordingly the lower right-hand corner of
the slide of view 4d is coated with only a monomolecular layer of
ovalbumin. The lower left-hand corner of view 4d is pronouncedly
darkened indicating the presence thereon of a bimolecular protein
layer comprising a first monomolecular layer of bovine serum
albumin, previously adsorbed thereon, having a second monomolecular
layer of rabbit anti-bodies to bovine serum albumin immunologically
complexed with the underlying bovine serum albumin. It will
accordingly be seen that only the specifically reacting protein
pair formed a bimolecular layer on the slide, all other coatings
thereon being monomolecular.
In a modification of this embodiment which provides for a medical
diagnostic system, the substrate having metal spheres thereon is
partially immersed, to a first depth, in a solution of a first
antigen. The substrate is coated in a monomolecular layer by the
first antigen over the area which is immersed in the solution. The
substrate is then dried and is more deeply immersed, to a second
depth, in a solution of a second antigen. The second antigen does
not adhere to the first antigen but does adhere to the uncoated
portion of the substrate which is immersed in the solution. The
substrate is then again dried and immersed in a solution of a third
antigen to a third and greater depth. The third antigen adheres to
that portion and only the portion of the substrate which is not
coated with the first or second antigen. The process is repeated
for any number of antigens of interest. The resulting product is a
substrate coated with a plurality of bands of monomecular layers of
different antigens. This coated slide is the diagnostic tool. The
medical diagnostic procedure is then to immerse the slide in a
specimen of, for example, blood for a period of typically several
hours in duration. The slide is then removed from the specimen and
after washing in water is viewed by reflected or transmitted light.
The slide then exhibits a pattern of lighter and darker bands which
are indicative of the antibodies present in the specimen from which
information a medical diagnosis is made.
FIGS. 2a, 2b and 2c are highly magnified elevation views of a
portion of diagnostic apparatus in accordance with the last
discussed embodiment of this invention. FIG. 2a shows a portion of
substrate material 31 having a plurality of globules of evaporated
metal 32 attached thereto. Particles 32 are preferably formed by
the evaporation of indium onto substrate 31 as discussed above but
may also be formed by evaporation of gold, silver, tin, lead, or
other metal having similar nonwetting and atomic mobility
characteristics. Any of a large number of metals will exhibit such
characteristics as the temperature of the substrate is varied.
After contact with a medium containing an antigen, the slide
segment comprising substrate 31 and metal globules 32 is partially
or completely coated with a monomolecular layer of molecules 34 of
the antigen as indicated in FIG. 2b generally at 33. The apparatus
indicated at 33 is the diagnostic instrument which is used to test
suspected solutions for the presence of corresponding specifically
reacting antibodies to antigen layer 34. If the apparatus indicated
generally at 33 is exposed to specifically reacting antibody to the
antigen layer 34, the apparatus will acquire the appearance
indicated generally in FIG. 2c at 35 in which substrate 31 and
metal globules 32 are coated with a bimolecular layer comprising
the layer 34 of the antigen forming a first monomolecular layer
overlying substrate 31 and globules 32 and a second monomolecular
layer consisting of the layer 36 of the specifically reacting
antibody to the first immunologically bonded to the molecules of
the first and overlying the first layer, the metal globules, and
the substrate.
FIG. 6 illustrates a modification of this embodiment of the
invention which provides for increased contrast in the coated
diagnostic slide. In FIG. 6, slide 31 having metal globules 32
applied thereto and coated with antigen monomolecular layer 34, as
in FIG. 2b, is immersed, the coated side downwardly, in a quantity
71 of light reflecting liquid. Slide 31 is then viewed from its
upper surface by reflected light. In order to provide for total
reflection of light incident on the diagnostic apparatus of FIG. 6,
reflecting liquid 71 is preferably a metallic liquid, and is more
particularly, preferably mercury. If a non-metallic liquid is
employed, the optical incidence and viewing angles employed in
using the FIG. 6 embodiment become critical if total reflection is
to be observed. The appearance of a test slide viewed in accordance
with these teachings is similar to that shown in FIG. 4, but with
enhanced contrast.
FIGS. 3a, 3b and 3c are highly magnified sectional elevational
views of apparatus useful for diagnostic purposes in accordance
with another embodiment of this invention. Indicated generally in
FIG. 3a at 40 is a substrate 41 coated with a monomolecular layer
of antibody molecules 42 which has been prepared as discussed
above. Indicated generally in FIG. 3b at 43 is substrate 41 and
antigen layer 42 to which has been immunologically bonded a second
monomolecular layer of molecules 44 in accordance with the
procedures discussed above. A second substrate 45 has thereon a
drop 46 of a reagent capable of cleaving immunological bonds, e.g.,
a weak acid solution. The mutually facing surfaces of substrates 41
and 45 having thereon respectively a bimolecular layer and a
reagent drop of, for example, citric acid in a 0.1 normal solution,
are then physically brought into contact with each other. In
accordance with an inventive discovery of this invention, the
reagent drop 46 severs the immunological bonds between molecules 42
and molecules 44 without affecting the bimolecular characteristics
of either antigen or antibody and without severing the adhesion
bond between the first molecules 42 and substrate 41. When
substrates 41 and 45 are again separated as indicated in FIG. 3c
generally at 47, substrate 41 has adhering thereon a monomolecular
layer of antigen molecules 42 and substrate 45 has adhering thereon
a monomolecular layer of corresponding specifically reacting
antibody molecules 44. Substrate 41 with molecules 42 thereon may
then be used again as a test slide in accordance with the other
previously discussed embodiment of this invention. Substrate 45
with specifically reacting antibody 44 ahdering thereto may also be
used as a test slide to test suspected solutions for the presence
of molecules of its corresponding antigen in accordance with the
other previously discussed embodiments of this invention. This
embodiment of this invention is considered to be particularly
significant because, as has already been noted, in the case of the
usual immunogens of biological interest, antigens are fairly
readily available in purified form and so may be absorbed onto a
substrate to form a test slide for the detection of the presence of
antibodies in suspected solutions but antibodies are not so
available. Therefore, previously to this invention it has generally
not been possible to produce test slides for testing suspected
solutions for the presence of antigens. The method and apparatus
illustrated in FIG. 3, however, provides for the production of test
slides comprising substrate 45 coated with a monomolecular layer
of, for example, antibody molecules 44 which may be used to test
suspected solutions for the presence of antigen.
Another embodiment of diagnostic apparatus in accordance with this
invention is illustrated structurally in FIG. 8, and its principle
of operation is illustrated graphically in FIG. 7. As illustrated
in FIG. 8, diagnostic apparatus in accordance with this invention
comprises a gold substrate 91, which, for reasons of economy, is
preferably a thin gold layer plated onto another metal, and which
has adsorbed thereon a monomolecular layer 92 of first antigen or
antibody. Gold has an absorption band within the visible spectrum.
This fact accounts for the characteristic color of gold and
provides for the operation of this embodiment of this
invention.
FIG. 7 illustrates the operation of this embodiment and constitutes
a set of reflectivity curves in which relative reflectivity is
plotted as a function of wavelength. Curve 93 represents the
reflectivity of gold metal. Curve 94 represents the reflectivity of
gold metal having a monomolecular antigen layer thereon. Curve 95
represents the reflectivity of gold metal having a bimolecular
layer thereon. Accordingly, a gold substrate such as 91 has, in
accordance with curve 93, the characteristic bright yellow color of
gold metal. When the first antigen layer 92 is applied to substrate
91, the appearance of the test slide, in accordance with curve 94,
has a dull yellow appearance. After the test slide has been exposed
to a solution suspected of containing corresponding immunologically
reacting antibody to the antigen comprising layer 92, the test
slide, if such specifically reacting antibody was present in the
solution, has a bimolecular layer thereon and a reflectivity
characteristic indicated by curve 95 which provides a distinctly
green appearance.
In tests which have been performed to date, it appears that the
embodiments of this invention which employ a substrate including
metal globules, that employing a gold substrate, and that employing
a metal, e.g., titanium, overlaid with an oxide coating, e.g., an
oxide of titanium, are the most generally useful. Furthermore, it
has been determined that these embodiments have differing
sensitivities as functions of the thicknesses of films of interest.
Specifically, the greatest sensitivity of the embodiment having a
substrate including metal globules occurs with films having
thicknesses below approximately 200 A. The gold and other substrate
embodiments have the greatest sensitivity for films exceeding 30 A
in thickness.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The following examples, which are to be regarded as illustrative
and not limiting, show how surface immunological tests are carried
out according to this invention.
EXAMPLE 1
An inhibition test for hepatitis is carried out as follows: Glass
slides are coated with a thin indium layer and overcoated with a
gold layer. The use of indium undercoating is required to improve
the adhesion between the glass and the gold. It is found further
that the diffusion of the indium into the overlying gold improves
the optical characteristics of the test slides. The slides thus
prepared are coated with a monomolecular layer of
hepatitis-associated antigend. Samples of human blood to be tested
for the presence of hepatitis are prepared by mixing therein a
quantity of antibodies to hepatitis-associated antigen sufficient
to be immunologically removed from the mixture if
hepatitis-associated antigen be present in the sample. The test
slides are then immersed in the previously prepared blood samples.
When removed, the slides which have been immersed in hepatitis
negative samples have a bimolecular protein layer thereon
comprising the hepatitis-associated antigen previously applied
thereto and a second layer of antibodies to hepatitis-associated
antigen immunologically bonded thereto evidenced by a greenish band
on the slide. Slides having been immersed in hepatitis positive
samples, on the other hand, retain their original dull yellow
appearance indicating that only the monomolecular
hepatitis-associated antigen layer is present thereon, the
antibodies which have been introduced into the sample having
complexed with the antigen therein and precipitated out of the
sample to be unavailable to react with the antigen on the test
slide.
EXAMPLE 2
In a direct test for hepatitis, glass-indium-gold slides are
prepared as described in Example 1 and then coated with a
monomolecular layer of antibodies to hepatitis-associated antigen.
Some of the slides are then immersed into pooled serum taken from
hepatitis patients and therefore known to contain
hepatitis-associated antigen. Others of the slides are immersed in
serum samples known to be free of hepatitis-associated antigen.
Those slides which have been immersed in the pooled serum have
bimolecular protein layers thereon, detectable visually as darker
zones as described above, while those immersed in the other blood
had only monomolecular layers thereon.
Theoretically, the direct test described in Example 2 is the
preferred diagnostic procedure both because of the relative
simplicity thereof and also because, in this case, the direct test
has a higher sensitivity than the inhibition test of Example 1. The
higher sensitivity of the direct test results from the fact that
hepatitis antigen molecules are very much larger than molecules of
antibody to hepatitis-associated antigen. Therefore, the direct
test involves a much larger percentage change in film thickness,
and accordingly greater contrast is observable.
The concentration of hepatitis-associated antigen in the blood of a
person having been exposed to the disease is a function of time.
The concentration of hepatitis-associated antigen is quite high
during the clinical and immediately preclinical phases of
hepatitis. In the far post-clinical phases, however, the
concentration of hepatitis-associated antigen in the person's blood
is very low. Both conditions are medically of interest. Detection
of the antigen during the clinical and immediately preclinical
phases is of value for the purposes of diagnosis. Detection of the
antigen in the far post-clinical phase is of value for screening
the blood of prospective blood donors. As a practical matter the
simpler, more sensitive direct test is preferable for diagnostic
purposes since it may be readily performed because of the
relatively high concentration of hepatitis-associated antigen in
the blood sample.
EXAMPLE 3
The inhibition test of Example 1 described for screening purposes
is evaluated experimentally to determine its value relative to
other known screening tests for hepatitis. In these tests,
hepatitis positive pooled serum is diluted with known hepatitis
negative serum. Using the inhibition procedure of Example 1, a
dilution of one part hepatitis positive pooled serum to 32 parts
known hepatitis negative serum results in reliable detection over a
one-hour period. Using a dilution of one part hepatitis positive
pooled serum to 3000 parts known negative serum, reliable detection
is obtained over a twenty-hour period. These results will be seen
to be very similar in sensitivity and time consumed, respectively,
to the presently known counterelectrophoresis procedure and
radioimmunoassay procedure. Therefore, a screening procedure in
accordance with this invention is seen to provide results
comparable to the best obtainable with prior art procedures, with
the additional advantage of obtaining the results through a
significantly less expensive procedure.
The use of a gold coated substrate in preference to a metal globule
coated substrate in the aforedescribed hepatitis diagnostic
procedure is preferred because hepatitis-associated antigen
molecule are approximately 210 A in diameter. Such film thicknesses
are well within the capability of gold coated substrates to provide
sensitive indication, but are too thick for high sensitivity of
indication using the metal globule coated substrate.
EXAMPLE 4
A diagnostic test for gonorrhea is carried out as follows: A glass
slide is metallized with a layer of titanium and the surface layer
is then oxidized to produce a cover layer of an oxide of titanium.
Neisseria gonorrhoeae extract is dissolved in salt water to produce
a concentration of 1 mg./ml. A drop of the solution of antigen is
placed in the center of the slide. The slide is incubated in a
moist chamber (plastic box filled with wet sponges) at room
temperature (23.degree. C.) until the antigen adheres to the oxide
surface (10-30 minutes). The slide is washed with distilled water
and blown dry with a jet of air. The slide can be stored at this
point for later use. To use, the test slide is immersed in blood
serum which contain antibody to the Neisseri antigen; the total
volume is 2 ml. The slide and antibody solution are incubated at
23.degree. C. in a plastic box attached to a shaker, for 12 hours.
After incubation, the slide is again washed with distilled water.
If, after drying, a sharply contrasting visible spot is observed in
the center of the slide, this indicates a positive reaction. In
cases where the blood serum does not contain gonorrhea antibodies,
no bimolecular layer will form, and no spot will be seen.
EXAMPLE 5
A diagnostic test for rubella is carried out by the general
procedure of Example 4. A gold metallized glass slide is used and
the antigen is an extract of a rubella viral culture. After
treatment of the slide with a human serum preparation containing
rubella antibody, a sharply contrasting visible spot is observed.
The procedure is repeated substituting a tantalum metallized glass
slide. Substantially the same results are obtained.
At present, rubella antibodies are detected using a
hemagglutination-inhibition (HAI) test. It is performed on patients
in obstetrics clinics as well as females of child-bearing age who
pass through health clinics. Such tests require two days; the test
of this invention requires two hours. Moreover, there is a high
correlation with HAI results.
EXAMPLE 6
A diagnostic test for poliomyelitis is carried out by the general
procedure of Example 4. A gold metallized glass slide is used and
the antigen is an extract of a polio viral culture. After treatment
of the slide with a human serum preparation containing polio
antibodies, a sharply contrasting visible spot is observed. The
procedure is repeated substituting a tantalum metallized glass
slide, with substantially the same results.
This test procedure is much simpler than the usual method in which
polio antibody level is determined by a complement-fixation
test.
EXAMPLE 7
A test is made for vesicular stomatitis proteins by the general
procedure of Example 4. A gold metallized glas slide is used and
the antigen is an extract of vesicular stomatitis virus. After
treatment of the slide with proteins specific to the antigen, a
sharply contrasting visible spot is observed. Tantalum metallized
glass slides can be substituted, and substantially the same results
are obtained.
EXAMPLE 8
A diagnostic test for tetanus is carried out by the general
procedure of Example 4. A gold metallized glass slide is used and
the antigen is applied as an aqueous extract of tetanus toxoid.
After treatment with a human serum preparation containing tetanus
toxoid antibodies, a sharply contrasting visible spot is observed.
The procedure is repeated substituting a tantalum metallized glass
slide, with substantially the same results.
EXAMPLE 9
A diagnostic test for diphtheria is carried out by the general
procedure of Example 4. A tantalum metallized glass slide is used
and the antigen is an aqueous extract of the diphtheria bacterial
toxin. After treatment with a human serum preparation containing
antibodies to diphtheria toxins, a sharply contrasting visible spot
is observed.
EXAMPLE 10
A diagnostic test for syphilis is carried out by the general
procedure of Example 4. A gold metallized glass slide is used and
the antigen is an aqueous suspension of Treponema pallidum. After
treatment with a human serum preparation containing antibodies to
syphilis, a sharply contrasting visible spot is observed. Tantalum
metallized glass slides can be substituted, and substantially the
same results are obtained.
EXAMPLES 11 AND 12
Diagnostic tests for fungal antibodies are carried out by the
general procedure of Example 4. A tantalum metallized glass slide
is used and the antigens, respectively, are aqueous suspensions of
the family Aspergillus and Candida. After treatment with a
preparation of human blood sera containing the corresponding fungal
antibodies, sharply contrasting visible spots are observed.
EXAMPLE 13
A diagnostic test for a parasite is carried out by the general
procedure of Example 4. A gold metallized glass slide is used and
the antigen is an aqueous extract of Toxoplasma gondii. After
treatment with a human serum preparation containing antibodies to
this parasite, a sharply contrasting visible spot is observed. The
procedure is repeated substituting tantalum and indium metallized
glass slides with substantially the same results.
Quantitative tests are also carried out on gold and tantalum
metallized glass slides, by making two-fold dilutions of the
patient's serum (antibody) and using as the endpoint of the
titration that concentration corresponding to that providing the
last visible spot.
The parasite has an unknown etiology but it has been determined
that latent and/or unsymptomatic infections in pregnant women cause
many birth defects. No other serological method has been developed
to test for antibodies to this organism.
EXAMPLE 14
A diagnostic test for antiobodies to human nucleoprotein-DNA is
carried out by the general procedure of Example 4. A gold
metallized glass slide is used and the antigen is an aqueous
suspension of the DNA-histone complex found in the nuclei of human
cells. After treatment with preparations of human blood sera
containing antibodies to the DNA-histone complex (nucleoprotein)
and/or antibodies to DNA (uncomplexed with histone), sharply
contrasting visible spots are observed. The advantage in this
technique is that both nucleoprotein and DNA antibodies can be
determined on a single slide. The procedure is repeated
substituting tantalum metallized glass slides with substantially
the same results.
In certain disease conditions, autoantibodies to these human
molecules can be directed to the DNA portion of the molecule to the
histone, or to the bond between the two. In 30% of the cases of
Lupus erythematosus, an antibody is produced which is specific to
DNA and as such its detection is diagnostically useful. Current
methods for testing for such antibodies involve a slide technique
using mouse liver tissue and a special peroxidase stain, or a
fluorescent antibody technique. Both procedures are long and the
slides are difficult to read. In contrast, the method of this
example is much shorter, the slides are easier to read, and they
show high correlations with the present methods of analysis.
EXAMPLE 15
A diagnostic test for antibodies to human protein thyroglobulin is
carried out by the general procedure of Example 4. Such antibodies
are formed in certain disease states, e.g., Hashimoto's disease. A
gold metallized glass slide s is used and the antigen is an aqueous
suspension of thyroglobulin. After treatment with a preparation of
human blood serum containing antibodies to thyroglobulin, a sharly
contrasting visible spot is observed. The procedure is repeated
substituting tantalum metallized glass slides with substantially
the same results.
EXAMPLE 16
A diagnostic test for antibodies to collagen is carried out by the
general procedure of Example 4. A tantalum metalized glass slide is
used and the antigen preparation is an aqueous medium containing
collagen. After treatment with rabbit blood sera containing
antibodies induced by administration of collagen fractions, a
sharply contrasting visible spot is observed. Rabbits fed high
cholesterol diets produce antibodies to collagen.
EXAMPLES 17 AND 18
An inhibition test is run to diagnose for pregnancy. A gold
metallized glass slide is used and the antigen is an aqueous
preparation of human chorionic gonadotrophin (HCG). Blood serum
containing HCG is serially diluted and a known concentration of HCG
antibody is added. The slide is immersed in the preparation. No
visible spot is detected if the test is positive. A visible spot
corresponds to a negative test. Levels of above 2000 International
Units of HCG/100 ml. of serum indicate pregnancy. The method can
also be adapted to test for tumors in prostate glands in males.
The procedure is repeated, substituting insulin for HCG. A blank
slide corresponds to a positive test. Antibody titrations are
successfully carried out on gold and tantalum-metallized glass
slides, with a sensitivity of 100 nanogram, which can be readily
increased.
EXAMPLE 19
A diagnostic test for antibodies to keyhole lymphet hemocyanin
(KLH) protein is carried out by the general procedure of Example 4.
A gold metallized glass slide is used and the antigen preparation
is an aqueous medium of KLH protein. After treatment with a
preparation of human blood serum containing antibodies to KLH
protein, a sharply contrasting visible spot is observed. The
procedure is repeated substituting tantalum metallized glass slides
with substantially the same results.
At presnt, research is being conducted to find an antigen that can
be used in humans to test the efficiency of their antibody response
systems. Necessary to this end is an antigen which has not
previously been encountered by the host, and KLH is a likely
candidate. The present method is useful in this work because it is
capable of detecting low levels of antibodies to KLH end is not
cumbersome, time-consuming and insensitive.
While this invention has been described with reference to
particular embodiments and examples, other modifications and
variations will occur to those skilled in the art, in view of the
above teachings.
Finally, although the reactive protein adsorbed on the substrate
surface has been described hereinabove as being an antigen, it
should be evident that such first layer reactive protein could be
an antibody, and the second layer protein would then be the antigen
specific to such antibody, and the third layer would again be the
antibody. Accordingly, it should be understood that within the
scope of the appended claims, the invention may be practiced
otherwise than is specifically described.
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